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Fermentation is a key technology in many fields and industries and is performed both on a mass production scale and on an experimental, bench top scale. For example, fermentation systems are used for the production of a large number of products such as antibiotics, vaccines, synthetic biopolymers, synthetic amino acids, and proteins. Fermentation technology is integral in the production of recombinant proteins using biological organisms, such as E. coli, and many other cell cultures. For example, production of commercial pharmaceuticals such as recombinant insulin (Eli Lilly), erythropoietin (Amgen), and interferon (Roche) all involve fermentation as an essential step.
In addition, the recent identification of the tens of thousands of genes comprising the human genome highlight an important use of fermentation, namely the production of the proteins encoded by those genes. The determination of each gene""s function is of paramount importance and therefore, the proteins encoded by those genes must be produced, e.g., by fermentation methods. Because each gene encodes at least one protein, tens of thousands of proteins must be produced and isolated. However, fermentation and isolation of the resulting protein products typically requires several labor intensive and time-consuming procedures. Fermentation systems that can produce tens of thousands of different proteins, e.g., in amounts sufficient for analysis are therefore needed. An additional advantage would be fermentation systems that are amenable to high throughput processes and the microtiter plate format used in many biotechnolgy applications.
Although, rapid advances in biotechnology have enabled the development of high throughput alternatives to traditional laboratory bench top processes, fermentation methods have not been amenable to automation. For example, limits in current fermentation technology prevent the uninterrupted processing flow that characterizes automated high throughput systems. Existing fermentation systems typically involve multiple handling steps by either a batch processing method or a continuous processing method.
Fermentations are typically carried out in batch mode or continuous mode. Batch mode processes are those in which a fermentor is filled with a medium in which cells are grown and the fermentation is allowed to proceed with the entire contents removed from the fermentor at the end for downstream or post-processing. The fermentor is then cleaned, re-filled, and inoculated for the fermentation process to be performed again. For example, current production scale batch processes involve first fermenting in large scale, bulk fermentation vessels, then processing the fermentation medium to isolate the desired fermentation product, followed by transferring this product into the production stream for further processing, and finally cleaning the fermentation apparatus for the next batch. In a large scale batch culture, it is generally necessary to provide a high initial concentration of nutrients in order to sustain cell growth over an extended time. As a result, substrate inhibition may occur in the early stages of cell growth and then may be followed by a nutrient deficiency in the late stages of fermentation. These disadvantages result in sub-optimal cell growth rates and fermentation yields. Another disadvantage of this method lies in the need to individually dispense the fermentation products from the bulk fermentation apparatus into separate sample vessels for further processing. Thus, by producing the fermentation product on a bulk scale, the fermentation product is not immediately available for automated processing. Further disadvantages include the decreased efficiency of both transferring the material to another sample vessel, as well as cleaning and sterilizing the fermentation apparatus for the next batch. These disadvantages result in increased production costs, inefficient production times and decreased yields.
Continuous batch processes involve siphoning off the fermentation product from the bulk fermentation vessel and continuously adding nutrients to the fermentation medium according to a calculated exponential growth curve. This curve, however, is merely an approximation that does not accurately predict cell growth in large, industrial scale quantities of fermentation medium. Consequently, due to the unpredictable nature of large scale fermentation environments, experienced personnel are required to monitor the feeding rate very closely. Changes in the fermentation environment may result in either poisoned fermentation products being siphoned off into the production stream or sub-optimal production yields due to starved fermentation mediums. As a further disadvantage, unpredictable fermentation product yields affect the accuracy of subsequent processing steps. For example, when the fermentation yield decreases, the amount of aspirating, the amount of reagent dispensed, or the centrifuge time is no longer optimized, or even predictable. Frequent or continuous monitoring of the fermentation process and adjustment of the fermentation conditions is often not practicable or efficient in a production scale process.
Neither of the current processes provides an efficient, automated production scale fermentation. However, fermentation remains a key processing step in a number of industries, particularly in biotechnology industries, and thus a need exists for incorporating fermentation processes into automated high throughput systems. A process that produces a precise, known, and repeatable amount of untainted fermentation product with limited human interaction or sample vessel transfer is essential to integrating fermentation into modern production processes. The present invention meets these as well as other needs that will be apparent upon review of the following detailed description and figures.
The present invention provides methods and apparatuses for simultaneously fermenting a plurality of samples, e.g., small samples in an 8 by 12 array. For example, the present invention provides a fermentation apparatus comprising a container frame configured to contain a plurality of sample vessels and a gas distribution arrangement coupled to the container frame. The fermentor provides for fermentation of large numbers of samples, e.g., to produce a large number of proteins. Alternatively, the fermentors of the invention provide a more efficient route for production scale fermentations.
In one aspect the invention provides a container frame configured to contain a plurality of sample vessels, e.g., in an array; and, a gas distribution arrangement configured to provide gas to a plurality of sample vessels, e.g., when the sample vessels are positioned in the container frame. The container frame is typically configured to contain an array of sample vessels, e.g., an 8 by 12 array, e.g., holding at least about 96, 384, or 1536 samples. The gas distribution arrangement typically comprises a gas inlet configured to deliver gas to a plurality of cannulas, which are configured to provide gas to the sample vessels.
In one embodiment, the container frame is a transportable container frame, e.g., configured for transport to a post-fermentation processing station. In addition, the container frame is optionally configured for placement within a temperature controlled area, e.g., water bath or a temperature controlled room, wherein a temperature controller is coupled to the container frame and/or to one or more sample vessels within the container frame.
In other embodiments, the container frame is autoclavable. For example, the container frame is autoclavable on its own or in combination with the gas distribution arrangement and/or the sample vessels.
The sample vessels typically comprise glass, plastic, metal, polycarbonate, ceramic, or the like. Each sample vessel typically has a volume of about 50 to 100 ml, e.g., and is used to hold a sample comprising less than about 80 mls, more typically about 65 mls. The samples in the plurality of sample vessels each have substantially the same composition or different compositions, e.g., to produce a large quantity of a single protein, or to produce multiple proteins simultaneously.
In other embodiments, the sample vessels optionally comprise a vent, e.g., for releasing built up pressure during fermentation. Sensors are also optionally placed in contact with one or more of the samples in the sample vessels, e.g., for monitoring temperature, pH, and the like.
In one embodiment, the gas distribution arrangement comprises a dispensing plate, an array of sample vessel areas, an array of cannulas, and a gas inlet. The dispensing plate typically comprises a top portion and a bottom portion that are joined together such that a hollow space exists between them. The array of sample vessel areas is typically located in a bottom surface of the bottom portion. Each sample vessel area comprises a recess and is positioned to correspond to the array of sample vessels. The array of cannulas are typically in fluid communication with the hollow space and protrude from a bottom surface of the dispensing plate through the sample vessel areas, e.g., to provide gas flow to the sample vessels. In some embodiments, the cannulas comprises a plurality of passages, e.g., at least three passages. The gas inlet is typically in fluid communication with the hollow space for delivering gas into a plurality of sample vessels via the cannulas during fermentation. For example, the gas distribution arrangement in some embodiments comprises a gas source that provides oxygen or a mixture of oxygen and at least one other gas to each sample vessel during operation of the apparatus.
In other embodiments, the gas distribution arrangement is optionally configured to allow delivery of one or more reagents to the sample vessels. For example, a dispenser is optionally coupled to the gas distribution arrangement, e.g., for dispensing one or more reagents into the plurality of sample vessels. The dispenser is typically configured to dispense reagents into the plurality of sample vessels, e.g., via a plurality of apertures corresponding to the sample vessels.
In addition, a process controller is operably coupled to the gas distribution arrangement, e.g., for controlling and/or monitoring gas flow to the plurality of sample being fermented.
In another aspect, the present invention provides a fermentor head for multiple sample fermentation. A typical fermentor head comprises a dispensing plate that comprises a top portion and a bottom portion, an array of sample vessels areas, an array of cannulas and a gas inlet. The bottom portion and the top portion of the dispensing plate are joined together such that a hollow space exists between them. The array of sample vessel areas is typically located in a bottom surface of the bottom portion of the dispensing plate, which sample vessel areas each comprise a recess and are positioned to correspond to an array of sample vessels. The array of cannulas are typically in fluid communication with the hollow space and protrude from a bottom surface of the dispensing plate through the sample vessel areas, e.g., 15 to 16 cm; with the gas inlet in fluid communication with the hollow space for delivering gas into a plurality of sample vessels via the cannulas during fermentation. Typically, the cannulas deliver gas adjacent to a bottom of the sample vessels. In some embodiments, the dispensing plate further comprises an array of apertures for accessing samples during fermentation. Alternatively, the cannulas are adapted to deliver gas, deliver fluid, or aspirate fluid from the sample vessels during fermentation. The vessels and samples used with the fermentor head typically correspond to those described above.
In another aspect, the present invention provides a method of fermenting a plurality of samples. The method typically comprises providing a plurality of sample vessels in a container frame, wherein each of the sample vessels contains a sample. The samples in the plurality of sample vessels are typically fermented, which fermenting comprises simultaneously delivering gas, e.g., oxygen, air, and/or, nitrogen, to each of the sample vessels via a plurality of cannulas associated with the sample vessels. Each sample typically has a volume of less than 100 ml, e.g., using sample vessels and a container frame as described above. In some embodiments, delivering gas to the samples comprises delivering air and oxygen to the samples over a period of time, during which period of time, the ratio of air to oxygen changes, e.g., linearly over time or in a stepwise manner over time.
In some embodiments, the methods further comprise detecting one or more fermentation conditions with a sensor coupled to one or more sample vessels and adjusting the fermentation conditions in the sample vessels, e.g., at pre-determined time intervals. For example, adjusting the fermentation conditions optionally comprises adding a feed solution to the sample vessels. Detecting optionally comprises measuring a pH of one of the samples; measuring a redox potential of one of the samples; measuring an optical density of one of the samples; and/or measuring a light emission from one of the samples.
In some embodiments, the methods further comprise pre-processing or post-processing the samples in the same set of sample vessels, e.g., in the same or a different location as the fermentation step. In some embodiments, the pre-processing and/or post-processing are performed robotically. Pre-processing and/or post-processing steps include, but are not limited to, centrifugation, aspiration, and/or dispensing of one or more reagent. For example, the methods optionally comprise transferring the sample vessels into a centrifuge rotor after fermentation or autoclaving the sample vessels, e.g., in the container frame prior to fermentation. In addition, the cannulas are also optionally autoclavable with the container frame.
In another aspect, the methods of the invention comprise positioning a plurality of sample vessels into a transportable container frame, which container frame maintains the sample vessels in an array. The plurality of samples is optionally placed into the plurality of sample vessels, e.g., before or after the vessels are positioned in the frame. A fermentor head is typically attached to the container frame, e.g., prior to or after the samples have been added to the vessels. The fermentor head typically comprises an array of cannulas, e.g., as described above. The cannulas typically correspond to the array of sample vessels and are inserted into the sample vessels when the fermentor head is attached. The samples in the sample vessels are then fermented, e.g., by simultaneously delivering a gas, e.g., oxygen, nitrogen, and/or air, to the samples via the array of cannulas. In some embodiments, the fermentation is an anaerobic fermentation comprising delivering an inert gas to maintain anaerobic fermentation conditions in the sample vessels. The methods optionally comprise robotic steps pre-processing steps, and/or post processing steps as described above. For example, the sample vessels and/or the sample container used in the above methods are optionally configured to be compatible with a centrifuge, wherein the method further comprises transporting the container frame and/or sample vessels to the centrifuge for centrifugation.
In some embodiments, the methods comprise transportation to an aspirator or dispenser, wherein the aspirator typically comprises an aspirator head which corresponds to the array of sample vessels within the container frame, in which case, the method further including operably attaching the aspirator head to the sample vessels and simultaneously aspirating the samples within the sample vessels. In other embodiments, a dispensing step is included, wherein the dispenser comprises a dispensing head corresponding to the array of sample vessels and the method further includes operably attaching the dispenser head to the sample vessels and simultaneously dispensing one or more materials into the sample vessels.
In another embodiment, the present invention provides a method of processing a plurality of fermentation samples. The method comprises fermenting a plurality of fermentation samples in a plurality of sample vessels, resulting in a plurality of fermented samples; robotically transporting the sample vessels containing the fermented samples to a centrifuge head; and centrifuging the fermented samples in the same sample vessels in which the fermentation was performed. For example, about 4 to about 10 sample vessels are optionally robotically transported to the centrifuge head at the same time. The method also optionally includes isolating a supernatant from the sample vessels after centrifuging the fermentation samples.